Summary More than 70 chemicals in tobacco smoke are carcinogens. Tobacco smoking is a risk factor for bladder cancer; however, despite many years of study, the principal chemicals in tobacco smoke and environment that damage DNA of the bladder are unknown. Aromatic amines (AAs) and heterocyclic aromatic amines (HAAs) arise in tobacco smoke and are responsible for much of the mutagenicity in urine of smokers. Some AAs (and possibly HAAs) are bladder carcinogens, and also induce liver, bladder, and colorectal cancer in rodents, and likely contribute to these cancers in humans. 4-Aminobiphenyl (4-ABP) is a human bladder carcinogen; however, several alkylanilines and structurally related HAAs occur in tobacco smoke at levels up to 100-fold greater than 4-ABP. Some epidemiological studies have linked N-nitroso compounds and polycyclic aromatic hydrocarbons as risk factors for bladder cancer. Robust measurements of DNA adducts are important to understand the chemicals in tobacco smoke, the environment, and diet that damage the bladder and may contribute to bladder cancer. Apart from 4-ABP, the chemicals in tobacco smoke that damage bladder DNA are unknown. The objective of this application is to apply robust screening tools to identify DNA adducts derived from exogenous and endogenous sources that damage bladder DNA. We will employ our newly developed mass spectrometry (MS) adductomic tools to identify the major chemicals in tobacco smoke condensate that form DNA adducts in the bladder of smokers. In Aim 1, we will conduct studies with cigarette smoke extract (CSE) and human bladder cells incubated alone or in co-culture with hepatocytes to assess the role of liver metabolism in DNA damage of the bladder. The panel of DNA adducts formed in bladder cells with CSE will serve as a guide to facilitate the characterization of the DNA adductome of the urothelium of smokers and nonsmokers undergoing bladder cancer surgery in Aim 2. Some procarcinogens in CSE can reach the bladder and undergo bioactivation by P450s expressed in the bladder, particularly aromatic amines and HAAs. Therefore, in Aim 3, we will examine the urinary exposome of AAs and HAAs in smokers participating in a tobacco cessation study by novel mass- tagging methods to measure the totality of these potential bladder carcinogens in urine, and assess the capacity of bladder enzymes to bioactivate these compounds. Our research is relevant to NIH's mission on public health. Our studies will provide a greater understanding about genotoxicants in tobacco smoke the environment that damage bladder DNA and contribute to bladder cancer. By merging chemical exposures and DNA adducts with mutational data, clues about the identities of environmental, dietary and endogenous genotoxicants can be established to identify subjects at risk for bladder cancer. Once identified, pragmatic measures can be taken to reduce human exposure to chemicals, by changes in life- style or mitigation of environmental exposures, which are probably the most efficient means of chemoprevention.